help: nucleus prep

Tao Wei twei at mccoy
Sat Jan 1 17:01:30 EST 1994


Hi, everybody out there!  

Recently I am preparing nuclei from tomato leaves to get nuclear extracts.   
As a start I followed a protocol from a friend. Leaf tissue is homogenized  
in chilled homogenization buffer (10 mM MES pH 6.8, 10 mM NaCl, 0.25 M  
sucrose, 5 mM EDTA, 0.2 mM PMSF, 0.15 mM spermine, 0.5 mM spermidine, 0.2%  
Triton X-100) (1:3 w/v), filterd through 2 layers and 6 layers of  
Miracloth respectively and then centrifuged at 2000x g for 10 min.  
Precipitate is resuspended in 10 ml of homogenization buffer and  
centrifuged again.  Initially this washing step can effectively separate  
nuclei from chloroplasts.  After I ran out of the homogenization buffer I  
had to prepare more following the recipe exactly the same as before.   
Unfortunately the newly-made buffer could not allow the separation of  
nuclei from chloroplasts, both nuclei and chloroplasts are precipitated. I  
tried different sucrose concentrations from 0.25, 0.5, 0.75, and 1.0 M,  
different pH from 6.0 to 7.0 and different salt concentrations from 10 to  
50 mM.  Even though I found pH is a factor of the most importance, no  
unique pH can work as well as the homogenization buffer (pH 6.8) used  
initially. It is so strange to me.  I do not have any idea about what is  
going on there.  
I also tried to used Percoll step gradient (10%, 30% and 60%, or 30%, 60%  
and 85%) to separate nuclei from chloroplasts.  In all these trials,  
chloroplast seems to be very heterogenous in density and distributed among  
all three gradients although most chloroplasts floated on top of percoll.  
I could not see any nuclear band. Could anybody out there have any  
suggestions or working protocols to help me?  



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